Bladder cancer (BC) is one of the most common malignancies in developed countries, ranking as the sixth most frequent neoplasm. The disease exists in two main forms: non-invasive BC, which lacks invasion into surrounding muscle tissue and is the more common form accounting for 75% of all cases, and muscle invasive BC, in which the tumor spreads into the urinary bladder muscle and may metastasize.
The gold standard for detection of BC is cystoscopy; however, this procedure is invasive, uncomfortable, costly and may provoke urinary tract infection. Moreover, cystoscopy may miss certain lesions, in particular small areas of carcinoma in situ. Currently, cytology is the only established non-invasive adjunct to cystoscopy. Although cytology is sensitive (70-80%) and highly specific (90-95%) for the diagnosis of high-grade BC, sensitivity is as low as 6-38% for detecting low-grade tumors (Bastacky et al., 1999).
In BC patients, urine is constantly in close contact with tumor cells and the urothelium surrounding them. Therefore it has been suggested that biomarkers in the urine or in tumor cells isolated from urine samples could be helpful for detecting and monitoring BC. Among the studied markers, several assays have been approved by the US FDA, including Bladder tumor antigen (BTA), BTA stat, Fibrin degradation products (FDP), Nuclear Matrix protein 22 (NMP-22), Immunocyt and FISH (Urovysion) (van Rhijn et al., 2005). Most of these assays manifest a higher overall sensitivity for BC compared to urine cytology, but their specificity is much less (van Rhijn et al., 2005): urinary tract infection, benign prostatic hypertrophy and renal calculi can affect these assays. Various other tests have been developed in the effort to identify biochemical markers that may have diagnostic and prognostic value, including tests to identify tumor-associated markers in the urine, serum, and bladder cancer tissue specimens. For example, urinary immunoglobulins have been found to increase in persons who have bladder cancer and appear to have some diagnostic and prognostic value. Other suggested markers are disclosed, for example, in U.S. Pat. No. 5,221,612, U.S. Pat. No. 7,332,290, U.S. Pat. No. 6,811,995 U.S. Pat. No. 6,280,956, U.S. Pat. No. 6,261,791, U.S. 20050196795, U.S. 20040126775, U.S. 20090136972 and WO 2004/0033641. To date, there is no consensus regarding the relevance of these tests and their role in enhancing or replacing cystoscopy.
The routine use of prostate-specific antigen (PSA) as a screening tool since the early 1990's has had a deep impact on early diagnosis of prostate cancer (CaP) and has resulted in an increase in CaP detection (McDavid et al., 2004). However, the use of PSA is currently being debated since it is not clear if PSA screening has led to a decline in mortality due to CaP (Andriole et al., 2009; Schroder et al., 2009). In addition, the vast amount of unnecessary biopsies due to false-positive PSA results places a large burden on the healthcare system and leads to patient discomfort (Damber et al., 2008). As a result, there is a need for more specific and more sensitive biomarkers for CaP.
Cancer is associated with local inflammation (Lin et al., 2007). Among the many known factors suggested to mediate or regulate various aspects of inflammatory reactions are heat shock proteins and cytokines.
Heat Shock Proteins (HSPs) are a class of functionally related proteins whose expression is increased when cells are exposed to elevated temperature or other stress (Lindquist et al., 1988). In neoplasms, the expression of HSPs is implicated in the regulation of apoptosis, as a modulator of p53, in the immune response against tumors, and in multidrug resistance (Kaufmann et al., 1990; Levine et al., 1991; Ciocca et al., 1993; Cappello et al., 2008), and HSP expression was found to be altered in certain tumors (Fuller et al., 1994). Recently, the expression of HSPs in tumor biopsy material particularly HSP60 and HSP90 were proposed as prognostic factors in BC. Using immunohistochemical staining, Lebert et al, showed that decreased expression of these HSPs in the tumor is correlated with invasive BC (Lebret et al., 2003). In addition, it was found that low HSP90 expression predicted failure of immunotherapy (Lebret et al., 2007).
Secretion of immunosuppressive cytokines is a non-specific strategy for tumor immune evasion in many malignancies. Thus far, studies addressing cytokine expression in bladder cancer focused on the immune response to Bacillus Calmette-Guérin (BCG) immunotherapy (Bohle et al., 1990; Fleischmann et al., 1989; Saint et al., 2001).
Certain studies report the detection of cytokines such as Interleukin (IL)-6, IL-8 and IL-10 in the urine of BC patients. Kochac et al. (2004) and Sheryka et al. (2003) suggest that urinary IL-8 levels are elevated in patients with invasive BC. These studies did not differentiate between newly diagnosed BC patients and patients that have been treated with intravesical or other anti-cancer therapy, known to affect urinary cytokine levels. Esuvaranathan et al. (1995), aiming to evaluate the effect of BCG treatment on urinary cytokine levels, report that urinary IL-6 levels are elevated in some of the BC patients. Cai et al. (2007) attempted to find an association between urinary levels of IL-6/IL-10 and recurrent BC. The authors indicated that no difference was found between the IL-6/IL-10 ratio of control subjects and of patients with initial BC.
Loskog et al (2007) demonstrated that bladder cancer tissue is infiltrated by regulatory T-cells expressing large amounts of TGF-β and IL-10 mRNA. They further confirmed that circulating T cells of these patients were unresponsive to polyclonal T cell activation compared to healthy controls. Helmy et al (2007), using immunelectromicroscopy, reported the expression of TGF-β protein in exfoliated malignant epithelial (urothelial) cells in the urine of patients with BC.
Cardillo and Ippoliti (2006) reported that IL-6, IL-10 and HSP90 immunoreactivity was higher in prostatic carcinoma (CaP) and intra-epithelial prostatic neoplasia than in normal prostatic tissue adjacent to neoplasia, and therefore, changes in their expression in human CaP samples could be used as a prognostic marker of disease progression.
IL-13 was originally described as a T cell-derived cytokine that inhibits inflammatory cytokine production (Minty et al., 1993; McKenzie et al., 1993; Punnonen et al., 1993) secreted from immune cells (Schmid-Grendelmeier et al., 2002; Brown et al., 1989). Though this original description remains accurate, the known functions of IL-13 have expanded over the past few years. In cancer, IL-13 inhibits CD8+ CTL-mediated tumor immunosurveillance (Terabe et al., 2000) and contributes to tumor escape from apoptosis and growth arrest (Skinnider et al., 2001; Kapp et al., 1999). Type I diabetic patients treated with HSP60 derived peptide, showed lesser need for exogenous insulin that was positively correlated with IL-13 production by T-cells, thereby indicating its importance as an anti-inflammatory mediator (Raz et al., 2001). A recent case-control study demonstrated a highly significant difference in mRNA and protein expression of IL-13 between patients with bladder cancer and controls (MalekZadeh et al., 2010; published after the priority date of the present invention).
To date, no cytokine or HSP urinary marker is known to differentiate between BC-afflicted subjects and subjects presenting with hematuria due to benign pathology, thus having adequate reliability to be used in clinical diagnosis. There remains an unmet need for improved compositions and methods for providing early diagnosis of genitourinary cancer such as bladder cancer or prostate cancer and for determining disease staging and prognosis.